marine drugs

Review Collagens of Poriferan Origin

Hermann Ehrlich 1,*, Marcin Wysokowski 2, Sonia Z˙ ółtowska-Aksamitowska 2, Iaroslav Petrenko 1 and Teofil Jesionowski 2 1 Institute of Experimental Physics, TU Bergakademie Freiberg, Leipziger str. 23, 09599 Freiberg, Germany; [email protected] 2 Institute of Chemical Technology and Engineering, Faculty of Chemical Technology, Poznan University of Technology, Berdychowo 4, 61131 Poznan, Poland; [email protected] (M.W.); [email protected] (S.Z.-A.);˙ teofi[email protected] (T.J.) * Correspondence: [email protected]; Tel.: +49-3731-39-2867

Received: 30 December 2017; Accepted: 28 February 2018; Published: 3 March 2018 Abstract: The biosynthesis, structural diversity, and functionality of collagens of sponge origin are still paradigms and causes of scientific controversy. This review has the ambitious goal of providing thorough and comprehensive coverage of poriferan collagens as a multifaceted topic with intriguing hypotheses and numerous challenging open questions. The structural diversity, chemistry, and biochemistry of collagens in sponges are analyzed and discussed here. Special attention is paid to spongins, collagen IV-related proteins, fibrillar collagens from demosponges, and collagens from glass sponge skeletal structures. The review also focuses on prospects and trends in applications of sponge collagens for technology, materials science and biomedicine.

Keywords: collagen; spongin; collagen-related proteins; sponges; scaffolds; biomaterials

1. Introduction Collagens constitute a superfamily of long-lived extracellular matrix structural proteins of fundamental evolutionary significance, found in both invertebrate and vertebrate taxa. They are among the most studied proteins due to their important functions in mammals, including humans. In addition to their structural function in cartilage and skin formation [1,2], as well as in the biomineralization of hard tissues [3] including bone [4] and dentine [5], collagens are involved in the regulation of diverse cellular functions and processes. During the last 60 years, research into collagens has evolved from the discovery of the structure of collagen [6,7], through studies on its chemistry and biochemistry [8–10], to present-day applications in cell therapy [11], biomedicine [12–14], cosmetics [15], and the food industry [16]. A rod-like triple-helical domain is the typical structural element in all collagens. However, they differ in their size, dislocations of the globular domains and imperfections within the triple helix, self-assembly behavior, and functional roles. The classification of collagens is based on structural and functional features of vertebrate collagens. For example, 28 collagen types have so far been identified and characterized at the molecular level in mammals (see for review [1,17]). Collagens are also divided into subfamilies based on their supramolecular assemblies: fibrils, beaded filaments, anchoring fibrils, and networks [11]. Usually, the amino acid sequences in collagens are responsible for the corresponding functional properties: energy storage capacity, stiffness, or elasticity [18]. Even the type of amino acid motif within the tropocollagen molecule of a collagen can significantly affect its mechanical properties. Consequently, it can be hypothesized that the diversity of collagen polyforms determines their future functions, even within the same organism. Marine vertebrate collagens have attracted scientific attention, mostly as products of fisheries [19]. In particular, fish-sourced collagens from skins and scales [20–22] have been studied and used as alternative collagen sources to avoid the potential risks associated with mammalian collagen due to bovine spongiform encephalopathy and the swine influenza crisis [23].

Mar. Drugs 2018, 16, 79; doi:10.3390/md16030079 www.mdpi.com/journal/marinedrugs Mar. Drugs 2018, 16, 79 2 of 21

In contrast to marine vertebrate collagens, similar structural proteins found in marine invertebrates represent one of the most ancient protein families within Metazoa. Marine invertebrate collagens arose earlier than their vertebrate analogs, and possess diverse unique structural features, including very special structure–function interrelations. Collagens from poriferans, coelenterates, annelids, mollusks, echinoderms, and crustaceans have been discussed in detail in several review papers (e.g., [24–32]) and books (e.g., [33,34]). The limiting factors that have hindered progress in this field of research are the difficulty of purifying marine invertebrate collagens and their relative species-dependent complexity. However, there are more than enough examples in practically every order of marine invertebrates to inspire experts in materials science and biomedicine, especially because the similarities in structure and biosynthesis between vertebrate and invertebrate collagens appear to be more impressive than the differences [24]. Sponges (Porifera) are the most simple and ancient multicellular organisms on our planet, and mostly live attached to a suitable substratum (rock, sandy sediments) on the seabed. Poriferans diverged from other Metazoans earlier in evolutionary history than any other known animal phylum, extant or extinct [35], with the first fossilized sponge remnants found in 1.8 billion-year-old sediments [36–41]. The phylum Porifera is divided into four classes: Hexactinellida, Demospongiae, and Homoscleromorpha, with silica-based skeletons; and Calcarea, with a skeletal network made of calcium carbonates [42]. According to Exposito et al. [27], before the divergence of the sponge and eumetazoan lineages took place, the genes which were responsible for the synthesis of some kind of ancestral fibrillar collagen arose at the dawn of the Metazoa. The duplication events leading to the formation of the A, B, and C clades of the fibrillar collagens occurred before the eumetazoan radiation. Interestingly, the similarity in the modular structure of sponges and humans is preserved only in the B clade of fibrillar collagens. This phenomenon correlates well with the hypothesis of the primordial function of type V/XI fibrillar collagens in initiating the formation of collagen fibrils [27]. Different systems of terminology relating to poriferan collagens are found in the literature, as sponges also display considerable polymorphism with respect to their collagenous structures. The insolubility of most poriferan collagens has been the main obstacle to carrying out any detailed biochemical analysis. Studies on the morphology and nanotopography of the collagenous fibrils have shown that they are dispersed throughout the intracellular matrix within the skeletons of sponges. Cuticular structures have been found in some sponges, but their molecular composition has not been determined [43]. It was accepted very early that collagen fibers in sponges can possess quite different morphological features [44]. Gross et al. isolated two distinct forms of collagen from Spongia graminea, which they called spongin A and spongin B [29]. The first corresponds to fine intercellular collagen fibrils, visible only by electron microscopy. The second, spongin B, forms macroscopically-visible rigid fibers which are characteristic of keratosan demosponges [43]. This was probably the moment when the terminological divergence arose with regard to the term spongin, which was initially proposed by Städeler [45] to denote the skeletal fibrous matter of bath sponges, and was also used for spongins A and B defined by Gross et al. [29]. Up to the present, the authors of numerous publications—especially those on applications of spongin-based scaffolds in tissue engineering [31,46–51]—have used the term collagen for spongin, or even defined spongin as “collagenic skeleton” [52]. Very recently, Tziveleka et al. [53] studied collagen from the marine demosponges Axinella cannabina and Suberites carnosus, and proposed three different terms: insoluble collagen (InSC), intercellular collagen (ICC), and spongin-like collagen (SlC). It is worth noting that the isolation of each form of collagen from demosponges is based on the selectivity of the method used. Data on collagen extraction methods from diverse mineralized sponges (Hexactinellida, Demospongiae) and sponges which lack mineralized skeletons (the subclass Keratosa)—including yields of the extracted collagens—may be found in the relevant papers. In this review, we focus on the structural diversity of collagens and collagen-like proteins in selected sponges, with particular focus on their origin, structural features, and applications in Mar. Drugs 2018, 16, 79 3 of 21 biomedicine and technology, including materials science and biomimetics. The review has the ambitious goal of providing thorough and comprehensive coverage of poriferan collagens (Figure1) as a multifacetedMar. Drugs topic 2018, with16, x controversial hypotheses and numerous open questions.3 We of 21 begin with a brief descriptionbiomedicine of spongins and technology, and their including practical materials applications. science and Next, biomimetics we examine. The review the collagen has the IV-related proteins in diverseambitious representatives goal of providing thorough of Porifera. and comprehensive Special attention coverage is of paid poriferan to Chondrosia collagens (Figuresp. 1) collagens and as a multifaceted topic with controversial hypotheses and numerous open questions. We begin with their applicationsa brief indescription marine of biotechnology, spongins and thei biomedicine,r practical applications. and cosmetics. Next, we examine Finally, the wecollagen discuss IV- the current state of workrelated related proteins to the in unique diverse representatives hydroxylated of collagen Porifera. Special discovered attention in is anchoring paid to Chondrosia siliceous sp. spicules of psychrophiliccollagens deep-sea and glass their applications sponges. Wein marine are optimistic biotechnology, that biomedicine both the, attemptsand cosmetics. to establishFinally, we implications discuss the current state of work related to the unique hydroxylated collagen discovered in anchoring for poriferansiliceous collagens spicules and of the psychrophilic numerous deep open-sea glass questions sponges. raised We are inoptimistic this review that both will the inspireattempts the scientific community toto carry establish out implications research for into poriferan collagens collagens and a collagen-relatednd the numerous open proteins questions from raised sponges, in this as ancient and intriguingreview structural will inspire biopolymers. the scientific community to carry out research into collagens and collagen-related proteins from sponges, as ancient and intriguing structural biopolymers.

Figure 1. Schematic overview of the collagens and collagen-like structural proteins of poriferan origin Figure 1. Schematicdescribed overviewin this review. of the collagens and collagen-like structural proteins of poriferan origin described in this review. 2. Spongins as Enigmatic Structural Proteins in Sponges

2. Spongins as EnigmaticIt is recognized Structural that so-called Proteins spongioblasts in Sponges—derived from the epithelium of sponges—are responsible for the formation of spongin. Minchin claims that the fibers of skeletal spongin are It is recognizedformed extracellularly; that so-called however, spongioblasts—derived the cuticular spongin fibrils arefrom of intracellular the epithelium origin [19]. of In sponges—are contrast to such structural proteins as collagen, fibroin (silk), elastin, resilin, and keratin, the responsible forchemistry the formation and molecular of spongin.biology—including Minchin the claimssequences that—of thespongins fibers so far of remain skeletal unknown. spongin It are formed extracellularly;seems however, that spongin the is cuticular the last enigmatic spongin proteinaceous fibrils are biopolymer, of intracellular although origin it is of [ 19very]. Inancient contrast to such structural proteinsorigin and as h collagen,as undergone fibroin more than (silk), 300 elastin,years of investigations. resilin, and Spongin keratin, in thethe form chemistry of cell-free and molecular skeletons of diverse bath sponges (Figures 2 and 3) has been used for more than 3000 years [54,55] biology—includingfor painting, the bathing sequences—of, and cleaning, spongins as padding for so battle far remain armor, for unknown. medical purposes, It seems and as thata vessel spongin is the last enigmaticfor proteinaceous drinking water [56]. biopolymer, A brief overview although of the practical it is of applications very ancient of spongin origin from and bath has sponges undergone more than 300 yearsin biomedicine of investigations. and technology Spongin in recent in times the is formgiven in of the cell-free next section. skeletons of diverse bath sponges A suggestion of a similarity between silk and bath sponge skeletal fibers was reported for the (Figures2 andfirst3) time has by been Geoffroy used in for1705 more [57], and than was 3000based yearson his chemical [ 54,55 experiments.] for painting, After that, bathing, attention and cleaning, as padding forwas battlepaid to practical armor, applications for medical of sponges purposes, in pharmacology and as adue vessel to the forpresence drinking of iodine water in their [56]. A brief overview of theskele practicaltons. For example, applications in 1819, ofAndrew spongin Fyfe— froma professor bath sponges of chemistry in in biomedicine Aberdeen—identified and technology in large quantities of iodine in the marine sponge Spongia usta, the “Coventry Remedy”, which was used recent times iseven given in ancient in the China next [58]. section. In 1841, bath sponges were described as those in which the essential base A suggestionof the skeleton of a similarity consists of keratose between fibrous silk matter. and At bath that time, sponge the structural skeletal and fiberschemical was similarity reported for the first time bybetween Geoffroy horny in fibers 1705 of [ 57sponges], and and was silk basedwas again on suggested his chemical by Croockewit experiments. [59]. It would After appear that, attention that the horny matter of sponge is closely analogous to silk and related proteins, differing from them was paid to practicalonly in that applicationsit contains additional of sponges halogens. According in pharmacology to Croockewit due, the tochemical the presence formula of horny of iodine in their skeletons. Formatter example, must be as in follows: 1819, 20(C Andrew39H62N12O Fyfe—a17) + I2S3P10 professor [59]. Schlossberg of chemistryer [60], however, in Aberdeen—identifiedreported the large quantitiesvery slight of iodine solubility in of the the fibrous marine matter sponge in ammoniacalSpongia solution usta, of the copper “Coventry hydroxide. Additionally, Remedy”, which was treatment with diluted sulfuric acid leads to the identification of leucine and glycocoll, in contrast to used even inthe ancient isolation China of tyrosine [58]. and In serine 1841, from bath sericin sponges under similar were conditions. described Städeler as those in 1859 in obtained which the essential base of the skeleton consists of keratose fibrous matter. At that time, the structural and chemical similarity between horny fibers of sponges and silk was again suggested by Croockewit [59]. It would appear that the horny matter of sponge is closely analogous to silk and related proteins, differing from them only in that it contains additional halogens. According to Croockewit, the chemical formula of horny matter must be as follows: 20(C39H62N12O17) + I2S3P10 [59]. Schlossberger [60], however, reported the very slight solubility of the fibrous matter in ammoniacal solution of copper hydroxide. Additionally, treatment with diluted sulfuric acid leads to the identification of leucine and glycocoll, in contrast to the isolation of tyrosine and serine from sericin under similar conditions. Städeler in 1859 Mar. Drugs 2018, 16, 79 4 of 21

Mar. Drugs 2018, 16, x 4 of 21 obtainedMar. Drugs similar 2018 results, 16, x [45] and introduced for the first time the scientific term spongin for this4 of 21 horny matter.similar Then, results in [45] 1864, and von introduced Kölliker for [61 the] carriedfirst time out the thescientific first histologicalterm spongin for studies this horny on sponges, matter. including investigationsThensimilar, in 1864,results of von the [45] Köllikerstructural and introduced [61] features carried for outthe of fibrousfirst the firsttime spongin. histological the scientific Diverse studies term iodine-containingspongin on sponges, for this including horny sponges matter. and theinvestigations matterThen, termedin 1864, of the as von “structuralJodospongin Kölliker features [61]”were carried of fibrous discussed out spongin. the by first HundeshagenDiverse histological iodine studies-containing in 1895 on [ 62sponges sponges,]. The and organic including origin of iodinetheinvestigations matter in bath termed sponge of as the “Jodospongin structural skeleton” were wasfeatures discussed suggested of fibrous by by Hundeshagen spongin. Harnack Diverse [ 63in ].1895 He iodine [62]. estimated The-containing organic the origin concentrationsponges and of of theiodine matter in bath termed sponge as “skeletonJodospongin was ”suggested were discussed by Harnack by Hundeshagen [63]. He estimated in 1895 the [62].concentration The organic of origin iodine in spongin at 1.1–1.2%, and demonstrated that superheated steam destroys the organic portion iodineof iodine in spongin in bath at 1.1sponge–1.2%, skeleton and demonstrated was suggested that superheated by Harnack steam [63]. destroysHe estimated the organic the concentration portion of of spongin fibers completely, liberating iodine. of iodinespongin in fibers spongin completely, at 1.1–1.2%, liberating and demonstrated iodine. that superheated steam destroys the organic portion of spongin fibers completely, liberating iodine.

FigureFigure 2. 2The. The mineral- mineral- and and cell cell-free-free skeleton skeleton of commercial of commercial HippospongiaHippospongia communis communisbath spongebath is an sponge is an example of a 3D spongin scaffold. example of a 3D spongin scaffold. Figure 2. The mineral- and cell-free skeleton of commercial Hippospongia communis bath sponge is an In 1898, Harnack isolated the “Jodspongin” and characterized it as an albuminoid-like product, example of a 3D spongin scaffold. containingIn 1898, over Harnack 8.5% iodine isolated and the9.4% “ nitrogenJodspongin [63].” In and 1926, characterized Clancey carried it out as ana critical albuminoid-like analysis of product, the literature to-date relating to the identification of spongin by other authors. In contrast to other containingIn over1898, 8.5%Harnack iodine isolated and the 9.4% “Jodspongin nitrogen” [and63]. characterized In 1926, Clancey it as an carried albuminoi outd a-like critical product, analysis physiologists, he suggested that the origin of the skeletal spongin in Euceratosa was not the same as of thecontaining literature over to-date 8.5% relating iodine and to the9.4% identification nitrogen [63]. ofIn 1926, spongin Clancey by other carried authors. out a critical In contrast analysis to of other that of the spongin which surrounds the spicules in the Pseudoceratosa [64]. At that time, the the literature to-date relating to the identification of spongin by other authors. In contrast to other physiologists,common bath he sponge suggested Hippospongia that the equina origin and of the the “Turkey skeletal cup spongin sponge” in Euspongia Euceratosa officinalis was notwere the the same as that of thespongesphysiologists, spongin most which studied he suggested surrounds with respect that the the spicules to origin spongin. inof the The skeletalPseudoceratosa results spongin published in [64 inEuceratosa ]. various At that papers was time, not [65,66] the the common same as bath spongeshowedthatHippospongia of remarkable the spongin equina differences, whichand surrounds duethe “Turkey to insufficiently the cup spicules sponge” effective in the Euspongia analytical Pseudoceratosa methods officinalis [64]. andwere At the thatthe use sponges of time, themost common bath sponge Hippospongia equina and the “Turkey cup sponge” Euspongia officinalis were the studiedcommercial with respectsponges tothat spongin. had been Thevariously results prepared published and bl ineached. various Consequently, papers [65 different,66] showed results remarkable sponges most studied with respect to spongin. The results published in various papers [65,66] differences,on the chemical due to nature insufficiently of spongins effective from particular analytical species methods were obtained. and the use of commercial sponges that showed remarkable differences, due to insufficiently effective analytical methods and the use of had beencommercial variously sponges prepared that had and been bleached. variously Consequently, prepared and bl differenteached. Consequently, results on the different chemical results nature of sponginson the from chemical particular nature species of spongins were from obtained. particular species were obtained.

Figure 3. Scaning electron microscopy (SEM) image of anastomosed spongin fibers from the demosponge H. communis, which are organized as sets of unconnected structures with dendritic architecture.

Figure 3. Scaning electron microscopy (SEM) image of anastomosed spongin fibers from the Figure 3. Scaning electron microscopy (SEM) image of anastomosed spongin fibers from demosponge H. communis, which are organized as sets of unconnected structures with dendritic the demosponge H. communis, which are organized as sets of unconnected structures with architecture. dendritic architecture.

Mar. Drugs 2018, 16, 79 5 of 21

For example, Clancey [64] isolated up to 7% of iodogorgonic acid besides the other amino acids in acidic hydrolysates of spongin. Clancey [64] did not identify hydroxyproline in spongin fibers of Hippospongia equina which had been treated with acid and alkali. It should be noted here that in natural collagen, a 3(S)-hydroxy-L-proline (3-Hyp) residue occurs together with a 4-Hyp residue, which is known to markedly increase the conformational stability of the collagen triple helix [67]. Hydroxyproline is found almost exclusively in collagen [8]. Thus, Clancey found a remarkably high amount of glutamic acid (18.4%), as well as 14% glycine, 5.7% proline, 2.8% tyrosine, 11% tryptophan or histidine, and a trace of cystine. Block and Bolling [68] presented the following results on the chemistry of spongin (Table1).

Table 1. Amino acid composition of spongin.

Constituent Content (%) Nitrogen 13.0–14.8 Sulfur 0.7 Iodine 0.84–1.46 Histidine 0–0.2 Lysine 3–3.6 Arginine 4.3–5.9 Cystine 2.8 Tyrosine 0–0.8 Tryptophan 0 Phenylalanine 3.3 Glycine 13.9–14.4 Diiodotyrosine 4.7 Molecular ratio of lysine to arginine 4:6

The content of glycine (about 14%) in this spongin is significantly lower than in collagen (between 25% and 33%) [8]. Thus, until the identification of two different spongins by Gross et al. in 1956, spongin was recognized, for the most part, as a halogenated scleroprotein (see Table2)[ 69–71] or neurokeratin-like protein [68] due to the presence of cystine.

Table 2. The chemistry of spongin according to Ackerman and Burkhard [69].

C H N I Br S Cl Ashes 47.00 6.28 16.06 1.41 2.93 0.87 0 1.16

Consequently, it is very curious that the two morphologically-distinct forms of spongin fibers—termed spongin A and spongin B—were classified by Gross et al. [29] as members of the collagen family. This was probably because such an analysis was supported by electron microscopy and X-ray diffraction, and by their general amino acid pattern, including corresponding glycine and hydroxyproline content. Ratios of glycine to hydroxyproline were 1.6 and 1.8 for spongins A and B, respectively. The results obtained with small-angle X-ray diffraction and electron microscopy showed the diameter of the spongin A unbranched fibril to be on the order of 20 nm, with an axial period of about 650 Å. The large and branched fibers of spongin B were 10–50 µm in width and composed primarily of bundles of thin unbranched filaments less than 10 nm wide [29]. Both fiber types and the amorphous matrix contain hexosamine, hexose, pentose, and uronic acid. Glucosamine, galactosamine, glucose, galactose, mannose, fucose, arabinose, and uronic acid were identified chromatographically in both spongin A and in the amorphous substance. It was shown that spongin B contains a small amount of amino sugar plus glucose and galactose. In contrast to mammalian collagen, neither spongin can be dissolved at all by collagenase (Clostridium hystolyticum) or pepsin, nor were they dissolved to any appreciable extent in alkali solutions or dilute acid [29]. In a paper by Katzmann et al. [72], it was reported that spongin B accounts for over 70% of the dry weight of the bath sponge H. gossypina, Mar. Drugs 2018, 16, 79 6 of 21 and contains approximately 7% by weight of glucosylgalactosylhydroxylysine but a negligible amount of other sugars. Recently,Mar. Drugs 2018 Langasco, 16, x et al. [52] isolated glycosaminoglycans (GAGs) from sponginous6 skeletons of 21 of selected bath sponges. Total GAG content—expressed as µg hexuronate/mg dry weight—shows bath sponge H. gossypina, and contains approximately 7% by weight of glucosylgalactosylhydroxylysine some variability among the tested species, being 0.171 ± 0.021, 0.367 ± 0.028, and 0.460 ± 0.081 for but a negligible amount of other sugars. H. communis Spongia officinalis S. lamella Recently,, Langasco et ,al. and [52] isolated, glycosaminoglycans respectively. The data (GAGs) obtained from suggestsponginous that skeletons these sponge GAGsof areselected structurally bath sponges. divergent Total from GAG vertebrate content—expressed GAGs [52 as]. μg hexuronate/mg dry weight—shows Thus,some variab it seemsility that among spongin the tested chemistry species, is being made 0.171 very ± 0.021, complex 0.367 by ± the0.028 presence, and 0.460 of ± diverse 0.081 for halogens H. (I, Br)communis which have, Spongia never officinalis been reported, and S. lamella in natural, respectively. collagens The or data keratins. obtained This suggest may explainthat these the sponge very high resistanceGAGs ofare this structurally proteinaceous divergent biopolymer from vertebrate to enzymatic GAGs [52]. treatment. Its unique resistance to various enzymes—includingThus, it seems amylases, that spongin lysozymes, chemistry is trypsin, made very pronase, complex collagenases, by the presence and of diverse other proteases—ishalogens well reported(I, Br) which [44 have,72]. never On the been other reported hand, in innatural the naturalcollagens environment or keratins. This diverse may explain bacteria the are very able to destroyhigh spongin resistance enzymatically of this proteinaceous and lead biopolymer to extremely to enzymatic high levels treatment. of damage Its unique to the resistance structure to of the various enzymes—including amylases, lysozymes, trypsin, pronase, collagenases, and other spongin-based skeletal fibers (see for details [73]). The isolation and purification of such special proteases—is well reported [44,72]. On the other hand, in the natural environment diverse bacteria “sponginasesare able” toremain destroy a challenge spongin enz forymatically future research, and lead and to extremelywill provide high a keylevels way of damage to obtain to peptides the that willstructure be useful of the for spongin detailed-based proteomic skeletal fibers analysis (see andfor details the sequencing [73]). The isolation of spongin. and purification of Understandingsuch special “sponginases of the nature” remain and a origin challenge of spongins—especiallyfor future research, and in will keratosan provide demosponges a key way to (the ordersobtain Verongiida, peptides Dictioceratida,that will be useful and for Dendroceratida)—changed detailed proteomic analysis and dramatically the sequencing after of the spongin. discovery of chitin as aUnderstanding second structural of the component nature and origin of the of skeletal spongins fibers—especially of demosponges in keratosan in demosponges the order Verongiida (the by theorders Ehrlich Verongiida, Group in Dictioceratida 2007 [74–76]., and It was Dendroceratida shown that)— anastomosingchanged dramatically and macroporous after the discovery skeletons of diverseof chitinverongiids as a second are made structural of some component kind of spongin–chitin of the skeletal biocomposites. fibers of demosponges The content in the of order chitin in suchVerongiida composites by ranges the Ehrlich between Group 10% in 2007 and [74 60%–76]. depending It was shown on that the anastomosing sponge species and [ 76macroporous]. The isolation skeletons of diverse verongiids are made of some kind of spongin–chitin biocomposites. The content and characterization of chitin in these composites was possible due to the well-known resistance of of chitin in such composites ranges between 10% and 60% depending on the sponge species [76]. The chitinisolation to dissolution and characterization in alkaline solutions of chitin [ 77 in– 79 these], in composites contrast to was spongins, possible which due to are the quickly well-known dissoluble in alkaliresistance [72,80 ].of Consequently, chitin to dissolution all publications in alkaline priorsolutions to 2007 [77– on79], spongins in contrast found to spongins, in Verongiida which spongesare mustquickly be re-examined. dissoluble in The alkali only [72,80]. existing Consequently, and up-to-date all publications classification prior ofto spongins2007 on spongins is that found proposed in by GarroneVerongiida in 1978 sponges [43]. He must states be re that-examined. the following The only typesexisting of and spongins up-to-date can classification be defined of and spongins discussed (see Figureis that proposed4). The firstby Garrone spongin in 1978 is to [43]. be He found states in that the the form following of spiculated types of spongins fibers. can These be defined structures are associatedand discussed with (see the Figure endogenous 4). The first mineralized spongin is to skeleton be found of in thethe sponge.form of spiculated It is also fib responsibleers. These for the formationstructures of are wide associated fibers which with the include endogenous only a mineralized very thin mineral skeleton element of the insponge. the core. It is This also kind responsible for the formation of wide fibers which include only a very thin mineral element in the of spongin is also resistant to mild acid or alkaline hydrolysis, as well as to pepsin and diverse core. This kind of spongin is also resistant to mild acid or alkaline hydrolysis, as well as to pepsin bacterial collagenases. However, this spongin can be partially destroyed by cuprammonium hydroxide and diverse bacterial collagenases. However, this spongin can be partially destroyed by treatmentcuprammonium at room temperature. hydroxide treatment at room temperature.

Figure 4. Diversity of spongins according to [43]. Figure 4. Diversity of spongins according to [43]. Second are the spongin fibers which form the skeleton of the keratosan demosponges: the Secondabundance are and the compactness spongin fibers of the which spongin form and thethe skeletonalmost complete of the lack keratosan of its own demosponges: inclusions— the abundancewhich andare replaced compactness with foreign of the sponginparticles— andtestify the almostto the originality complete of lack theof spongin its own in inclusions—which this group. A are replacedtypical example with foreign of such particles—testify spongin can be found to the in originalitythe genus Ircinia of the, characterized spongin in thisby spongin group. fibers A typical

Mar. Drugs 2018, 16, 79 7 of 21 example of such spongin can be found in the genus Ircinia, characterized by spongin fibers cored with foreign debris (sand microparticles) [81]. Recently, Castritsi-Catharios et al. [82–84] described the chemical elements and the physical properties of such skeletal spongin from diverse commercial sponges before and after chemical treatment. The importance of the so-called basal spongin is evident for all sponges as sessile animals. In sponges with no organized internal skeleton, the organism is attached to the substratum by a more or less continuous layer of external spongin. This spongin is secreted by the basopinacocytes. The basal spongin is continuous with the internal spongin only in poriferans with an organized skeleton, formed either of spongin fibers or spiculated fibers. Due to the function of the basal spongin in such demosponges as Chondrosia reniformis (a species lacking spicules and internal spongin), the animal is attached strongly to its substratum. The basal spongin is discontinuous in erect sponges, where it forms the starting points of the internal organized skeleton. However, in the endemic fresh water demosponge Lubomirskia baicalensis, the holdfast which is responsible for attaching the sponge body to the hard substratum contains both basal spongin and chitin [77]. The extremely flexible and elastic organic structures which are morphologically similar to mineralized spicules are known as spiculoids [85]. They have been described in representatives of the genera Darwinella and Igernella (order Dendroceratida), where they are either free or partly joined to the fibers of the skeleton. They are compressible and can be easily torn apart. Finally, spongin may be responsible for the protection of gemmule shells. Gemmules are formed within the tissues of most freshwater and some marine sponges, and represent morphologically diverse asexual reproductive spherical bodies a few tenths of a millimeter to more than 1 mm in diameter, composed of a dense mass of identical cells and surrounded by an organic coat called the shell. The shell of gemmules is fortified with siliceous spicules and gemmoscleres, which are embedded into a matrix composed of both chitin and a collagenous protein. This collagen has been referred to as spongin [43].

Trends in the Applications of Spongins The history of studies on the chemistry, molecular biology, biochemistry, and bioinspired materials science of spongins remains relevant today, partly due to the poorly understood basis of ecological disaster in the case of sponge diseases, but mostly due to recent progress in the direct applications of sponge skeletons as 3D spongin scaffolds in tissue engineering and biomimetics. Additionally, the marine ranching of bath sponges worldwide is a crucial factor in the adoption of spongins as renewable naturally prestructured proteinaceous scaffolds. The spongin-based skeletons of bath sponges appear to possess a number of unique and useful properties, which had been exploited long before such scientific fields as tissue engineering and bioengineering were proposed. As reviewed by Szatkowski et al. [86], from the 18th century commercial bath sponges were valued in medicine due to their softness, high compressive strength, ability to retain shape, and high sorption rates. For these reasons, they were used as compression bandages for pressing open sinuses, in overcoming strictures of body passages (including the rectum), for dilation of the cervix uteri [86–90], and in the form of sponge tents applied in the uterus to expand the cavity and enable examination. More intriguingly, fragments of sponge skeleton were used as small prostheses in early “plastic surgery” [91]. Revolutionary results were obtained by Hamilton in 1881. In a paper entitled “On sponge-grafting”[92], he reported the following case. A woman underwent surgery for removal of a mammary tumor, during which a large area of skin was removed. The skin was replaced with a thin slice of an aseptic sponge skeleton, which ten days after the surgery was observed to be vascular, and three months later was covered with epithelial tissue (Figure5). Mar. Drugs 2018, 16, 79 8 of 21 Mar. Drugs 2018, 16, x 8 of 21

FigureFigure 5. Sketch5. Sketch of of a fragmenta fragment of of spongin spongin framework framework ((bb)) surroundedsurrounded by by a a great great number number of of living living cells cells (a,c()a in,c) ain sponge-grafting a sponge-grafting application application (adapted (adapted from from [ 92[92]).]).

Today, spongin-based scaffolds are actively used in diverse applications related to tissue Today, spongin-based scaffolds are actively used in diverse applications related to tissue engineering. Positive results have been reported with human osteoprogenitor cells on the skeleton of engineering.S. officinalis Positive [46], with results osteoblast have been-like MG reported-63 cells with growing human on osteoprogenitor spongin from Hymeniacidon cells on the skeletonsinapium of S. officinalis[93] and with[46], mouse with osteoblast-like primarily osteoblasts MG-63 on cells spongin growing from on Callyspongiidae spongin from marineHymeniacidon demosponges sinapium [49].[93 ] andRecently, with mouse Nandi primarily et al. [51] osteoblastshave proposed on sponginthat the skeleton from Callyspongiidae of the marine marinesponge Biemna demosponges sp.—alone [49 ]. Recently,and in Nandi combination et al. [51 ] with have proposedgrowth factors that the—is skeleton a promising of the marine biomaterial sponge forBiemna bonesp.—alone repair and and in combinationbone augmentation. with growth factors—is a promising biomaterial for bone repair and bone augmentation. BesidesBesides applications applications in in the the biomedical biomedical field, field, spongin-based spongin-based scaffolds have have been been successfully successfully usedused as adsorbentsas adsorbents of of diverse diverse dyes dyes [ 94[94,95],95] and and as as supportssupports forfor enzymeenzyme immobiliza immobilizationtion [96]. [96 ].It Itwas was recentlyrecently shown shown that that spongins spongins are are thermostable thermostable up up to to 260260 ◦°CC[ [86,97,98].86,97,98]. This This property property opens opens the the door door forfor applications applications of spongin-basedof spongin-based scaffolds scaffolds with with 3D 3D architecture architecture in in such such novel novel scientific scientific disciplines disciplines as Extremeas Extreme Biomimetics Biomimetics [99], [99], with with the aimthe aim of developing of developing novel novel advanced advanced composite composite materials. materials.

3. Collagen3. Collagen IV IV and and Related Related Proteins Proteins in in Sponges Sponges It isIt nowis now well well established established that that collagens collagens are are keykey toto thethe structuralstructural integrity integrity and and biomechanical biomechanical propertiesproperties of variousof various tissues tissues of of Metazoans. Metazoans. One One ofof them,them, thethe basementbasement membrane membrane-forming-forming collagen collagen IV,IV, is extremely is extremely ancient. ancient. Collagen Collagen IV IV networks networks have have aa polygonalpolygonal architecture that that endows endows basement basement membranesmembranes (BMs) (BMs) with with a tensile a tensile strength strength sufficient sufficient to protect to protect tissues tissues from mechanical from mechanical stress, instress, addition in to servingaddition as to important serving as regulatorsimportant of regulators the dynamic of the events dynamic associated events associated with cell withadhesion, cell adhesion, signaling, andsignaling, survival [and100 ].survival According [100]. to According the modern to viewthe modern [101], onlyview the[101] presence, only the of presence the collagen of the IV collagen gene was IV gene was precisely correlated with the emergence of BMs in animals. Thus, the triple helical precisely correlated with the emergence of BMs in animals. Thus, the triple helical collagen IV was collagen IV was required for the development of BMs. required for the development of BMs. BMs underlie the epithelia in Metazoa from sponges to humans [102]. Interestingly, until 1996, BMs underlie the epithelia in Metazoa from sponges to humans [102]. Interestingly, until 1996, basement membrane structures and type IV collagen were known to be present in all multicellular basement membrane structures and type IV collagen were known to be present in all multicellular animal species except sponges. In Porifera, BMs are associated with the basal surfaces of polarized animalepithelial species cells except [103]. sponges.After the Infirst Porifera, report on BMs the are identification associated of with type the IV basal collagenous surfaces sequences of polarized in epithelialthe homoscleromorph cells [103]. After sponge the first Pseudocorticium report on the jarr identificationei by cDNA and of genomic type IV collagenousDNA [103], this sequences collagen in thehas homoscleromorph been found in diverse sponge poriferans.Pseudocorticium For example, jarrei by incDNA corresponding and genomic transcriptome DNA [103], data this fromcollagen a hascalcareous been found sponge in diverse (Sycon poriferans.coactum) and For another example, homoscleromorph in corresponding sponge transcriptome (Corticium candelabrum data from), a calcareoustwo new sponge type IV ( Syconcollagen coactum genes) were and anotherfound in homoscleromorpheach [104]. Homologs sponge of important (Corticium components candelabrum of ), two new type IV collagen genes were found in each [104]. Homologs of important components

Mar. Drugs 2018, 16, 79 9 of 21 of basement membrane genes, including type IV collagen, have been found in the Demospongiae Spongilla lacustris, Ircinia fasciculata, and Chondrilla nucula [105]. The discovery of type IV collagen in Calcarea and Demospongiae is very important, because nowhere in this group has a BM-like structure been noted. The presence of type IV collagen in glass sponges (Hexactinellida) remains to be detected. Polyclonal antibodies have detected type I (but not type IV) collagen in the anchoring spicules of the Hyalonema sieboldii glass sponge [3]. The relationship between type IV collagen and the so-called spongin short chain collagen (SSCC) [106] is still under investigation [101]. SSCC has been considered as ancestral to type IV collagen [107]. Like type IV collagen, SSCC also has NC1 domains which produce the globular heads particular to type IV collagen and which are required for assembly of the unique scaffold of the BM (see for review [104]). It is suggested that collagen IV and its spongin variant are primordial components of the extracellular microenvironment, where collagen IV especially was a key player in the evolution of epithelial tissues in Metazoa, including sponges, due to the transition to multicellularity [101]. Interestingly, collagen IV from the demosponge Chondrosia reniformis has recently been patented as a source of special membranes for biomedical applications [108]. The collagen was isolated with an extraction solution of 100 mM Tris-HCl, 10mM EDTA, 8 M urea, and 100 mM 2-mercaptoethanol, rendering the protein in the form of a precipitate. This was used for the development of stable and non-cytotoxic type IV collagen membranes, which can be applied in tissue engineering and regenerative medicine approaches for epithelial repair, regeneration, or replacement. The technology includes the re-epithelialization of any single and stratified epithelium, with emphasis on the skin.

4. Fibrillar Collagens in the Mesohyl of Demosponges The mesohyl includes a noncellular colloidal mesoglea with embedded collagen fibers, spicules, and various cells, being as such a type of mesenchyme. It is currently debated whether the mesohyl and pinacoderm layers in sponges are true tissues [109]. Collagens serve several functions in sponges [27,106,110]. The formation of mesohyl certainly involves the activity of fine fibrils made of fibrillar collagen. The collagen fibrils both mediate cell–matrix interactions via membrane receptors and provide the structure of the extracellular matrix (ECM), a situation observed in vertebrates. The increase in the structural diversity of fibrillar collagen chains, their different forms of maturation, and interactions with other ECM components appeared during the process of evolution [111]. The diversity of sponges which contain high amounts of fibrillar collagen within their mesohyl has been described previously (see for review [110–112]). Fibrillar bundles, formed by the association of several hundred collagen fibrils, have been observed in diverse species of Tethya, Chondrosia, Chondrilla, Jaspis, and Suberites (see for details [112,113]). The densely packed bundles of collagen fibrils are secreted exclusively by the highly polarized lophocyte cells [43,111]. These are actively moving cells, pulling behind them a bundle of regularly arranged collagen fibrils. Another kind of collagen-producing cell has been discovered in the mesohyl of the demosponge Suberites domuncula [114,115], in which the expression of collagen genes is controlled by silicate and myotrophin [116]. SEM observations have revealed the complex collagen network surrounding the spicules within the mesohyl of adult specimens (Figure6). Collagen fibers have also been identified in the mesohyl of the demosponge Haliclona rosea [116]. Collagen has also been reported in the mesohyl of such Calcarea sponges as Leucosolenia sp. and Leucandra sp. [117]. Collagen fibril content is also high in the external asexual buds that occur in Tethya lyncurium [118]. Similarly, the buds of T. sychellensis contain a dense collagen matrix [119]. Buds consist of cellular masses that sprout out from the surface of adults and are able to develop into new functional individuals [119]. Recently, special attention has been focused on fibrillar collagens in the mesohyl of C. reniformis. This species is the only sponge which has been experimentally proven to contain a dynamic collagenous Mar. Drugs 2018, 16, 79 10 of 21

Mar.mesohyl Drugs 2018 capable, 16, x of stiffening upon being manipulated [120]. It was shown that the different10 of 21 physiological states recorded in laboratory experiments are expressions of the mechanical adaptability adaptabilityof the collagenous of the mesohylcollagenous of C. mesohyl reniformis of, andC. reniformis suggest that, and stiffness suggest variability that stiffness in this variability sponge is in under this spongecellular is control under [cellular121]. control [121].

FigureFigure 6 6.. SEMSEM view view through through the the collagenous collagenous mesohyl mesohyl of of the the demosponge demosponge S.S. domuncula domuncula. .Layers Layers of of collagencollagen fibrils fibrils (A,B (A,B) )are are a a result result of of the the activity activity of of the the unique unique collagen collagen-producing-producing cells cells which which are are seen seen toto line line up up along along the the surface surface of of the the spicules spicules (C (C––EE).). The The line line of of ce cellslls (A (A) )can can move move from from left left to to right right along along thethe spicule, spicule, depositing depositing a a rough, rough, nanofibrillar nanofibrillar collagenous collagenous layer layer in in their their wake wake ( (CC)) (see (see also also [114]). [114]).

5. Chondrosia Collagens 5. Chondrosia Collagens Collagens from the demosponge Chondrosia reniformis (Nardo 1847) have received attention from Collagens from the demosponge Chondrosia reniformis (Nardo 1847) have received attention from researchers since 1970 [113,117–119] due to their diversity (type IV collagen, fibrillar and nonfibrillar researchers since 1970 [113,117–119] due to their diversity (type IV collagen, fibrillar and nonfibrillar collagens) [120] and interesting structural [121], physicochemical [122], and ecophysiological collagens) [120] and interesting structural [121], physicochemical [122], and ecophysiological properties [123–127]. For example, slices of fibrillar collagen incubated with collagenase are not properties [123–127]. For example, slices of fibrillar collagen incubated with collagenase are not modified even after 48 h of incubation, and do not show any changes in the aspect, consistency, or modified even after 48 h of incubation, and do not show any changes in the aspect, consistency, or fine fine structure of the fibrils. No kind of enzymatic damage was observed by electron microscope on structure of the fibrils. No kind of enzymatic damage was observed by electron microscope on the the isolated collagen fibrils after collagenase treatment. isolated collagen fibrils after collagenase treatment. The mechanical properties of this collagen have been partially described by Garrone et al. [117]. The cortex of Chondrosia sponges is less resistant than calf skin, but has mechanical properties of the same order as those of bovine nasal cartilage (Young’s modulus 150–250 kg/cm2 and 100–250 kg/cm2 respectively). Probably due to special mechanical features, the body of Chondrosia can slowly become flat and slide to avoid compression or stretch itself into a slender thread under continuous stress.

Mar. Drugs 2018, 16, 79 11 of 21

The mechanical properties of this collagen have been partially described by Garrone et al. [117]. The cortex of Chondrosia sponges is less resistant than calf skin, but has mechanical properties of the same order as those of bovine nasal cartilage (Young’s modulus 150–250 kg/cm2 and 100–250 kg/cm2 respectively).Mar. Drugs 2018 Probably, 16, x due to special mechanical features, the body of Chondrosia can slowly become11 of 21 flat and slide to avoid compression or stretch itself into a slender thread under continuous stress. SuchSuchcreeping creepingbehavior behavior of of a fibrousa fibrous and and living living material material provides provides a remarkable a remarkable example example for thefor studythe study of mechanicalof mechanical stresses stresses as morphogenetic as morphogenetic factors factors [117 [117].]. Although Although the the nanomorphology nanomorphology of ofC. C. reniformis reniformis collagencollagen fibrils fibrils has has now now beenbeen well investigated investigated (Figure (Figure 7)7)[ [121],121 ],there there is still is still a lack a lack of knowledge of knowledge about aboutthe relationshipthe relationship between between the the ultrastructural ultrastructural features features of of this this collagen collagen and its its mechanical mechanical and and physicochemicalphysicochemical properties. properties.

Figure 7. Schematic diagram of C. reniformis collagen fiber with numerous nanofibrils with Figure 7. Schematic diagram of C. reniformis collagen fiber with numerous nanofibrils with characteristic nanotopography.characteristic nanotopography. Along the fibril, Along one characteristically the fibril, one characteristically thick segment (protrusion) thick segment about (protrusion) 28 nm in diameterabout 28 is nm followed in diameter by two is equalfollowed thinner by two and equal closer thinner conjoined andsegments closer conjoined (interband) segments about (in 20terband) nm in diameter.about 20 The nm average in diameter. distance The between average the distance protrusions between is about the 67–69 protrusions nm. The is distance about 67 between–69 nm. two The consecutivedistance between peaks of two the interbandconsecutive regions peaks or of between the interband a protrusion regions and or anbetween adjacent a interbandprotrusion region and an isadjacent about 21–23 interband nm. The region average is about step height21–23 nm. between The average the protrusions step height and between the interband the protrusions regions was and calculatedthe interband to be regions about 4 was nm (seecalculated for review to be [121 about]). 4 nm (see for review [121]).

The well-known biocompatibility of C. reniformis fibrillar collagen has stimulated many studies The well-known biocompatibility of C. reniformis fibrillar collagen has stimulated many studies on on its possible applications in cosmetics and pharmacology (see for review [31]), including in its possible applications in cosmetics and pharmacology (see for review [31]), including in transdermal transdermal drug delivery [128]. drug delivery [128]. The production and selection of a triple transformed Pichia pastoris yeast strain expressing a The production and selection of a triple transformed Pichia pastoris yeast strain expressing stable P4H tetramer derived from C. reniformis sponge and a hydroxylated nonfibrillar procollagen a stable P4H tetramer derived from C. reniformis sponge and a hydroxylated nonfibrillar procollagen polypeptide from the same organism have recently been reported by Giovine et al. [118]. The polypeptide from the same organism have recently been reported by Giovine et al. [118]. The obtained obtained recombinant sponge P4H has the ability to hydroxylate its natural substrate in both X and recombinant sponge P4H has the ability to hydroxylate its natural substrate in both X and Y positions Y positions in the Xaa-Yaa-Gly collagenous triplets. It is suggested that the Pichia system could be in the Xaa-Yaa-Gly collagenous triplets. It is suggested that the Pichia system could be used for the used for the large-scale production of hydroxylated sponge- or marine-derived collagen large-scale production of hydroxylated sponge- or marine-derived collagen polypeptides, which have polypeptides, which have high pharmacological potential [118]. high pharmacological potential [118]. The possibility of the application of Chondrosia fibrillar collagen as an organic template for in The possibility of the application of Chondrosia fibrillar collagen as an organic template for vitro silicification has been confirmed in several studies [121,129,130]. There are no doubts that the in vitro silicification has been confirmed in several studies [121,129,130]. There are no doubts that the mechanical properties of biomimetically-inspired hybrid composites can be significantly improved mechanical properties of biomimetically-inspired hybrid composites can be significantly improved with the presence of this special collagen. with the presence of this special collagen.

6. Glass Sponge Collagen Collagen is known as a universal template in biomineralization, including both calcification and silicification. It is proposed that this biopolymer functions as a fundamental template in biomineralization, inasmuch as it is very ancient from an evolutionary point of view and is common to many species and biological systems with a global distribution [131]. The identification of diverse

Mar. Drugs 2018, 16, x 12 of 21

6. Glass Sponge Collagen Collagen is known as a universal template in biomineralization, including both calcification and silicification. It is proposed that this biopolymer functions as a fundamental template in Mar. Drugs 2018, 16, 79 12 of 21 biomineralization, inasmuch as it is very ancient from an evolutionary point of view and is common to many species and biological systems with a global distribution [131]. The identification of diverse collagens in demosponges as described above suggests that they may also be found within skeletal structures in the sistersister group,group, thethe glassglass sponges.sponges. Hexactinellida Schmidt (Porifera), with more than 700 species, consists exclusively of marine glass sponges. These are psychrophilic organisms which can produce hugehuge biosilica-basedbiosilica-based skeletonsskeletons andand anchoringanchoring spiculesspicules atat temperaturestemperatures betweenbetween− −2 ◦°CC and 4 ◦°CC[ [132].132]. The challenging task of isolating and identifying collagen in the skeletal structures of diverse glass sponges was completed successfully only in 2010 [[3],3], following numerous attempts at gentle demineralization [133,134]. [133,134]. Stu Studiesdies in in this this area area have been motivated by the great flexibility flexibility of the glassy spicules, spicules, which which allows allows researchers researchers to tie to a tie spicule a spicule into a bundleinto a bundle(Figure 8 (Figure). It has been 8). It suggested has been thatsuggested this peculiar that this feature peculiar of spiculesfeature of in spicules the hexactinellids in the hexactinellids must be due must to thebe due presence to the of presence a structural of a carcassstructural of organiccarcass of nature organic both nature on the both surface on the (Figure surface9) and (Figure within 9) and the spiculeswithin the [133 spicules]. [133].

Figure 8 8.. PhotographPhotograph demonstrating demonstrating the the unique unique flexibility flexibility of the of theH. sieboldiH. sieboldi anchoringanchoring spicule, spicule, and andschematic schematic view view of the of the role role of ofspecial special hy hydroxylateddroxylated collagen collagen in in silica silica condensation condensation in in this this natural basilica structure (for details see [[3]).3]).

The organic phase has been identified as a highly hydroxylated fibrillar collagen which contains The organic phase has been identified as a highly hydroxylated fibrillar collagen which contains an unusual [Gly–3Hyp–4Hyp] motif predisposed for silica precipitation, and provides a novel an unusual [Gly–3Hyp–4Hyp] motif predisposed for silica precipitation, and provides a novel template template for biosilicification in nature [3]. This collagen presents a layer of hydroxyl groups that can for biosilicification in nature [3]. This collagen presents a layer of hydroxyl groups that can undergo undergo condensation reactions with silicic acid molecules with consequent loss of water. As a result, condensation reactions with silicic acid molecules with consequent loss of water. As a result, the initial the initial layer of condensed silicic acid will be held fixed to the collagenous template in a geometric layer of condensed silicic acid will be held fixed to the collagenous template in a geometric arrangement arrangement that will favor further polymerization of silicic acid. It therefore appears that collagen that will favor further polymerization of silicic acid. It therefore appears that collagen was a novel was a novel template for biosilicification that emerged at an early stage during metazoan evolution, template for biosilicification that emerged at an early stage during metazoan evolution, and that the and that the occurrence of additional trans-3-Hyp plays a key role in stabilizing silicic acid molecules occurrence of additional trans-3-Hyp plays a key role in stabilizing silicic acid molecules and initiating and initiating the precipitation of silica. the precipitation of silica. Collagen has also been reported as the main organic component of the spicules of the glass sponge Monorhaphis sp. [135] (Figure 10). Results of the amino acid analysis of protein extracts isolated from demineralized spicules of this sponge showed an amino acid content typical for collagens of the same origin. Comparison with the Microsatellite Database (MSDB) protein database led to the identification of alpha 1 collagen in two high-MW bands. In contrast to its analog in H. sieboldi, collagen isolated Mar. Drugs 2018, 16, x 13 of 21

Mar. Drugs 2018, 16, 79 13 of 21 and identified from Monorhaphis sp. was matched only to the type I collagen pre-pro-alpha (I) chain

(COL1A1)Mar. Drugs from2018, 16 dog, x (AAD34619) (MW 139,74) [135]. 13 of 21

Figure 9. SEM image of the nanofibrillar collagenous layer on the surface of an H. sieboldi glass sponge anchoring spicule.

Collagen has also been reported as the main organic component of the spicules of the glass sponge Monorhaphis sp. [135] (Figure 10). Results of the amino acid analysis of protein extracts isolated from demineralized spicules of this sponge showed an amino acid content typical for collagens of the same origin. Comparison with the Microsatellite Database (MSDB) protein database Figure 9. SEM image of the nanofibrillar collagenous layer on the surface of an H. sieboldi glass sponge led toFigure the identification 9. SEM image ofof the alpha nanofibrillar 1 collagen collagenous in two layerhigh- onMW the bands. surface ofIn ancontrastH. sieboldi to glassits analog sponge in H. anchoring spicule. sieboldianchoring, collagen spicule. isolated and identified from Monorhaphis sp. was matched only to the type I collagen pre-proCollagen-alpha (I) has chain also (COL1A1) been reported from asdog the (AAD34619) main organic (MW component 139,74) [135]. of the spicules of the glass sponge Monorhaphis sp. [135] (Figure 10). Results of the amino acid analysis of protein extracts isolated from demineralized spicules of this sponge showed an amino acid content typical for collagens of the same origin. Comparison with the Microsatellite Database (MSDB) protein database led to the identification of alpha 1 collagen in two high-MW bands. In contrast to its analog in H. sieboldi, collagen isolated and identified from Monorhaphis sp. was matched only to the type I collagen pre-pro-alpha (I) chain (COL1A1) from dog (AAD34619) (MW 139,74) [135].

Figure 10 10.. HighHigh-resolution-resolution transmiss transmissionion electron microscope image of a fragment of M. chuni collagen nanofibrilnanofibril isolatedisolated fromfrom thethe glassyglassy spicule spicule (for (for details details see see [135 [135]).]). The The nanomorphology nanomorphology of of such such fibrils fibrils is similaris similar to thatto that from fromH. sieboldiH. sieboldiglass glass sponge sponge collagen collagen [3], but[3], different but different from thefrom striated the striated collagen collagen fibrils fromfibrils the from demosponge the demospongeC. reniformis C. reniformis[121]. [121].

Figure 10. High-resolution transmission electron microscope image of a fragment of M. chuni collagen nanofibril isolated from the glassy spicule (for details see [135]). The nanomorphology of such fibrils is similar to that from H. sieboldi glass sponge collagen [3], but different from the striated collagen fibrils from the demosponge C. reniformis [121].

Mar. Drugs 2018, 16, 79 14 of 21

The existence of naturally occurring collagen–silica-based composites in the form of spicules of glass sponges stimulated material scientists to develop analogous hybrid materials. Due to the limited available amounts of glass sponge collagen for the development of silica-based composite materials, fibrillar collagen from the demosponge C. reniformis has been successfully used as an alternative by the Ehrlich research group [121,129]. More recently, a new concept in biosilica material synthesis which does not require phosphate supplements and is based on the fusion of stabilized polysilicic acid into a fluidic precursor phase upon infiltration into polyamine-enriched collagen has been proposed by the Tay research group [136–138]. It has recently been shown that silicified collagen scaffolds produced by infiltrating collagen matrices with intrafibrillar amorphous silica exhibit angiogenic and osteogenic potential and can be used in tissue engineering [139]. In work by Aime et al. [140], collagen triple helices have been confined on the surface of sulfonate-modified silica particles in a controlled manner. This gives rise to hybrid building blocks with well-defined surface potentials and dimensions. Additionally, oligomeric collagen-fibril matrices with tunable microstructural properties have been used to template and direct the formation of biocompatible mesoporous sol–gel silica to develop nanostructured hybrid organic–inorganic composites [141]. It was experimentally confirmed that silica mineralization kinetics are critical for the precision-tuning of properties of the hybrid materials, including porous microstructure, mechanical strength, depth of silica penetration, and mass transport properties. It has also been shown that microstructural properties of the collagen-fibril template are preserved in the silica surface of hybrid materials [142]. Such novel silica-collagen hybrid materials may be useful, for example, in the regeneration of bone tissue or in cellular microencapsulation [141].

7. Conclusions We have presented here only a brief discussion of selected examples, which nonetheless provide novel data concerning poriferan collagens. In spite of the progress made in this research field, numerous open questions remain. For example, additional investigations are necessary to obtain understanding of the nature and origin of halogenated spongins. It is still not clear how many collagen and/or keratin domains they contain. Additionally, the unique resistance of these biopolymers against diverse chemicals and enzymes remains poorly investigated. The possible role of collagens in the spiculogenesis of demosponges and formation of axial filaments must also be researched. The discovery of crystalline proteins within amorphous biosilica-based structures in sponges is ground-breaking in the understanding of biomineralization. What can be discovered about the crystallinity of collagen within biosilica-related structures in sponges? The existence of collagen-based crystals within siliceous biominerals could revolutionize our understanding of the origin and evolution of collagens, from the point of view of biomineralization in sponges as the first multicellular organisms on Earth. Further, the relationship of collagen and chitin in the skeletal structures of diverse sponge classes and orders is entirely unknown. Consequently, we believe that the use of modern X-ray imaging techniques based on the “diffraction before destruction” principle is the best way forward to gain understanding of the principles of the unique organization of collagen within both fossil and recent collagen-based biomineralized constructs. Novel approaches must be proposed which will bring together modern bioanalytical and molecular biology methods for better understanding of the fundamental principles of collagen fibrillogenesis and the mechanisms of its cross-linking in sponges, as well as details of the structural organization of poriferan collagens at the molecular and atomic levels. The best way to address this challenging task on these levels is by coherent synergetic collaboration using explicit reasoning and well-tested explanatory principles of multidisciplinary knowledge, experience, and new technologies. Finally, we suggest that studying the processes of marine farming of the collagen-producing demosponges has implications for a variety of practical large-scale applications, ranging from the design of highly effective extraction techniques to the development of novel collagen-containing composites for biomedicine and technology. Mar. Drugs 2018, 16, 79 15 of 21

Acknowledgments: This work was partially supported by DFG Project HE 394/3-2 and PUT Research Grant no. 03/32/DSPB/0806. M.W. is grateful for financial support from the Foundation for Polish Science: START 097.2017, and S.Z.-A.˙ for support from the DAAD and Erasmus Plus programs. Author Contributions: Hermann Ehrlich and Marcin Wysokowski researched the literature and wrote the manuscript; Iaroslav Petrenko prepared diagrams and images and edited the manuscript; Soni Z˙ ółtowska-Aksamitowska and Teofil Jesionowski discussed ideas and edited the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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